Gene knock-out (KO) is a genetic technique in which one or several of an organism’s genes are silenced (loss of function) by genome editing technologies. Developed in the 1980s, gene knock-out has become a useful way to study gene function. The basic principle for gene knock-out involves the introduction of specific double-strand breaks (DSBs) at desired locations in the genome, which are followed by the endogenous DNA homologous recombination repair (HR) or non-homologous end joining repair (NHEJ). There are currently three commonly engineered nucleases for introduction of DSBs: Zinc-finger nucleases, TALEN and Cas9 nuclease in the CRISPR/Cas9 system.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) gene editing technology is a revolutionizing tool developed in recent years. Bacteria use CRISPRs naturally as an anti-viral defense immunity mechanism. In that natural context, CRISPR DNA sequences are transcribed into crRNA (CRISPR RNAs) that in turn base pair with tracrRNA (trans-activating RNA). This tracrRNA/crRNA complex then leads to the destruction of specific viral DNA sequences via the Cas9 nuclease. In the man-made CRISPR/Cas9 system, tracrRNA and crRNA are packaged together to form a single-guide RNA (sgRNA or gRNA) that can guide the Cas9 enzyme to cut at a specific genome locus. So simply by designing functional gRNAs targeting different gene locus, the genome can be cut at any corresponding locations, which makes the cloning work much easier for this system in comparison with the other two technologies. Besides, the adaptability of CRISPR/Cas9 system widens its potential applications in many different ways.
All the superior advantages of CRISPR/Cas9 technology makes it a priority for genome editing in various organisms ranging from single cell organisms to mammals.
Cas9 nuclease is guided by the designed gRNA molecule to cut at a specific gene locus. The resulting DSB will then be spontaneously repaired by the cellular NEHJ repairing system, which leads to DNA insertion or deletion (indel) at the repairing site and the following inactivation of a functional gene.
SyngenTech provides one-stop service for generation of gene knock-out cell lines or model animals using CRISPR/Cas9 technology, including the upstream gRNA design and CRISPR/Cas9 vector construction to the downstream genotyping and breeding to homozygosis. You can also outsource parts of this complex process to us. We will offer you custom and top-quality services at unbeatable prices.
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